Due to its constant exposure to light, the ocular lens continuously generates reactive oxygen species (ROS), which if not quenched, initiate lipid peroxidation. Our studies have shown that unlike the whole lens, the lens epithelium and especially the human lens epithelial cells (HLEC) are rich in polyunsaturated fatty acids (PUFA). Under oxidative stress, these tissues generate lipid-derived aldehydes (LDAs) such as 4-hydroxynonenal (HNE) which are known to propagate the ROS-induced toxicity. The lens has efficient detoxification systems to detoxify the LDAs under physiological conditions. However, under prolonged oxidative stress the detoxification capacity of the lens for HNE is impaired. Our studies demonstrate that the oxidative pathway of HNE metabolism, catalyzed by ALDH, is crucial in maintaining the lens clarity under oxidative stress. Inhibitors of ALDH reduced the HNE oxidation and accelerated the oxidation-induced opacification in rat lens. Our studies further suggest that ALDH1 isozyme, present mainly in the epithelium, is the main catalyst of HNE oxidation to HNA. Since ablation of ALDH1 in the HLEC by antisense of ALDH1 increased HNE- and H2O2-induced toxicity (protein-HNE adducts and apoptosis) as compared to the scrambled antisense-transfected cells, we hypothesize that oxidation of HNE is essential to maintain the lens transparency under oxidative conditions and that ALDH1 is the main catalyst of HNE oxidation. Our aims are: (i) To ablate the ALDH1 in HLEC and rat lens by siRNA-ALDH1 and study the impairment of 3H-HNE oxidation, and oxidation-induced increase in protein-HNE, apoptosis and lens opacification. (ii) To investigate the kinetic properties of the lens ALDH1, using ALDH1 purified from HLEC and human lens recombinant enzyme and study the modification of the purified enzyme by thiol-modifiers, such as S-nitrosoglutathione, oxidized glutathione and hydrogen peroxide. (iii) To investigate ALDH1 modification and its role in animal models of oxidative cataract. (iv) To investigate the modification of ALDH1 in vitro by NO donors (4a) and in vivo by increasing the aqueous humor NO levels (4b) and study if NO synthase inhibitor prevents the modification of ALDH1 and toxicity in oxidation-induced cataract (4c). Our studies in this grant will thus (i) delineate the role of ALDH1 in the detoxification of LDA and (ii) demonstrate the mechanism of regulation of ALDH1 under oxidative stress.